This invention relates to the treatment of pain and the identification of targets for pain treatment.
The sensation of pain can be caused by damage to sensory nerves (neuropathy), as well as damage to non-neural tissues. Pain that results from neuropathy is often referred to the deafferented regions of the body surface (Davar and Maciewicz, Neurol. Clin. 7: 289-304, 1989). In humans, painful peripheral neuropathy is often persistent, disabling and resistant to treatment with analgesic drugs including morphine (Sweet, Neurosurg. 15: 928-932, 1984).
Painful neuropathy often results from the direct effects of diseases, such as diabetes, varicella zoster virus (shingles) infection, trauma, autoimmune disorders, and connective tissue diseases. Alternatively, pain can result as a consequence of disease treatment, as is often observed in patients with AIDS and cancer who receive chemotherapy, radiation therapy or surgery.
One candidate mediator of pain associated with nerve injury is the inflammation response (Hafer-Macko et al., Ann. Neurol. 39: 625-635, 1995; Swartz et al., J. Neuropathol. 15:9-10, 1995; Schmidt et al., Muscle and Nerve 19: 474-487, 1996). The inflammation response in the nerve includes local edema, disruption of the perineurium, Wallerian degeneration, and infiltration of the endoneurium by progressively increasing numbers of leukocytes (Sommer et al., J. Neuropathol. Exp. Neurol. 52: 223-233, 1993; Sommer et al., J. Neuropathol. Exp. Neurol 54: 635-643, 1995). Such signs of inflammation are well developed following trauma to the rat sciatic nerve (Avellino et al., Exp. Neurol. 136: 183-194, 1995; Dahlin, Neurosci. Lett. 184: 5-8, 1992; Danielsen et al., Biomaterials 14: 1180-1185, 1993; Monaco et al., J. Neurocytol. 21: 623-634, 1992; Perry et al., J. Exp. Med. 165: 1218-1223, 1987).
Another potential mediator of nerve pain is the potent vasoconstrictor peptide, endothelin-1 (ET-1) (Hickey et al., Am. J. Physiol. 248: C550-C556, 1985; Yanagisawa et al., Nature 332: 411-415, 1988). Administration of epinephrine (xe2x89xa710 nM) is known to reduce sciatic endoneurial blood flow to a degree similar to that observed with similar concentrations of ET-1 (Zochodne et al., Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1806-H1810, 1992). The intraperitoneal administration of ET-1 produces an abdominal writhing response in rodents that is ET-1 receptor-mediated. This response may be behavioral evidence of acute pain (Raffa et al., Exp. Ther. 276: 647-651, 1996a; Raffa et al., J. Pharmacol. Exp. Ther. 278: 1-7, 1996b). In humans, the intra-arterial administration of ET-1 is reported to induce severe pain that is associated with prolonged touch-evoked allodynia in the injected limb (Dahlof et al., J. Hypertension 8: 811-818, 1990). The ETA subtype receptor-dependent effects are G-protein coupled and may be linked to L-type calcium channel signaling as observed in other tissues (Reynolds et al., Biochem. Biophys. Res. Commun. 160: 868-873, 1989). ET-1 is secreted in high concentrations by metastatic prostate cancer cells (Nelson et al., Nature Medicine 1(9): 944-949, 1995).
Cancer pain affects over 75% of patients with metastatic cancer. Thus, over 1 million patients in the United States and well over 9 million patients worldwide suffer cancer pain (Bonica, The Management of Pain, second edition, ed: J. J. Bonica, pages 400-460, 1990). The pain is often debilitating and difficult to treat, especially in patients with advanced disease. In patients with metastatic cancer of the prostate, pain treatment often requires very large doses of either systemic or intraspinal opioids, often an insufficient pain treatment that produces undesirable side-effects (Cherny and Portenoy, Textbook of Pain, third edition, eds: P. D. Wall and R. Melzack, pages 787-823, 1994). Clearly, a better understanding of the mechanisms that mediate the sensation of pain associated with neuropathy and cancer pain will lead to new target identification and the development of more beneficial pain treatments.
The invention provides a method of determining whether a compound alleviates vasoconstriction-independent nerve pain mediated by endothelin-1 (ET-1). The method involves (i) determining whether the compound has the ability to inhibit a vasoconstriction-independent ET-1 action, and, if the compound has the inhibitory ability, then ii) determining whether the compound reduces vasoconstriction-independent nerve pain by testing the compound in human patients suffering from pain mediated by the vasoconstriction-independent ET-1 action.
The method of determining whether the compound has the ability to inhibit a vasoconstriction-independent ET-1 action can involve an in vitro assay composed of steps such as the following: (i) layering functional ET or ETA receptors, e.g. in samples of neuronal or vascular cells or neuronal or vascular cell membrane preparations, onto culture plates, e.g., scintillator-coated 96-well plates, in paired samples that contain or lack the compound; (ii) adding ET receptor ligand, e.g. [25I]ET-1, measure specific binding, and assess ability of compound to inhibit ligand binding; if inhibitory compounds are identified, then (iii) further characterizing the agonist or antagonist effect of the compound on the activation of intracellular signaling, e.g. on G-protein coupled signaling, in cell-based assays; if ligands with antagonist or partial antagonist activity are identified, then (iv) screening compounds in an in vivo model, e.g., the direct sciatic nerve ET-1 application model, and assessing the effects on ET-1 induced pain behavior, e.g., hindpaw flinching.
Preferably, the compound inhibits the ET-1 action at a nerve ET receptor. Most preferably, the compound inhibits the ET-1 action at a nerve ETA receptor.
The invention also includes a method of treating a human patient to alleviate pain caused by vasoconstriction-independent ET-1 action. The method involves administering an effective amount of an ET inhibitor, e.g. an inhibitor from the group of sulfisoxazole, TBC-11251, BQ-123(American Peptide Co., Sunnyvale, Calif.; Clinalfa AG, Switzerland), PD 156707 (Parke Davis, Morris Plains, N.J.), A-127722 (Abbott Laboratories, Abbott Park, Ill.), LU 135252 (Knoll Pharmaceutical Co., Mount Olive, N.J.), BQ-788 (American Peptides Co., Sunnyvale, Calif.), bosentan (Hoffman LaRoche, Nutley, N.J.), TAK-044 (Takeda Chemical Industries Ltd., Osaka, Japan), SB 209670 (Smith Klein Beecham Pharmaceuticals, Philadelphia, Pa.), and SB 217242 (Smith Klein Beecham Pharmaceuticals, Philadelphia, Pa.) as described in Ann. Reports in Medicinal Chemistry, Section II, Ch. 7, Endothelin Inhibitors, pages 61-70, 1997 (ed. A. M. Doherty, Academic Press, Inc.).
The structures of the aforementioned ET-1 antagonists are provided below. 
Preferably, the ET inhibitor is an ETA-selective inhibitor from the group of TBC-11251, BQ-123, PD156707, A-127722, and LU 135252, and the compound inhibis ET-1 action at a ETA receptor on the nerve.
The method of treatment of the invention can be used to alleviate pain in a patient with a condition associated with increased ET-1 levels and for the treatment of diseases such as metastatic prostate cancer, metastatic breast cancer, sickle cell anemia, and other painful vascular diseases, e.g., migraine headaches, Reynaud""s disease, and peripheral vascular disease.
The invention also includes a method of determining whether a compound alleviates pain caused by nerve injury in human patients. The method involves (i) determining whether the compound has the ability to inhibit an inflammatory leukocyte response, and, if the compound has the inhibitory ability, then (ii) testing the compound in human patients suffering from pain caused by nerve injury to determine whether the compound alleviates the pain.
The method of determining whether the compound has the ability to inhibit an inflammatory leukocyte response can by carried out by conducting an in vitro assay, such as (i) harvesting leukocytes, e.g. rat leukocytes, and (ii) placing leukocytes in wells of a culture plate, e.g., a radially-distributed agarose culture plate, in paired samples which either contain or lack the compound, adjacent to wells containing minced nerve samples, e.g. minced rat sciatic nerve, (iii) incubating the plates for sufficient time, e.g. 8-12 hours, to allow leukocyte migration toward the nerve sample well, (iv) fixing and staining the leukocytes, and (v) quantifying the degree of leukocyte migration to assess the ability of the compound to inhibit leukocyte migration.
The invention also provides a method of treating a human patient to alleviate pain caused by nerve injury. The method involves administering an effective amount of a leukocyte migration inhibitor, e.g., itraconazole, mepaerine, ketoconazole, chloroquine, hydroxychloroquine, dipyridamole, or piroxicam (Feldene(copyright), Pfizer, Inc., New York, N.Y.).
One advantage of the present invention is the identification of specific, novel targets to use for developing new pain treatments. Given these targets, candidate compounds never before considered as effective pain treatments can be analyzed. For example, using these new targets, screening assays can be performed to identify new candidate compounds previously missed in other anti-pain screens. In addition, compounds that have previously been identified as affecting the targets of the invention, i.e., compounds identified as ET inhibitors and leukocyte migration inhibitors, can be reanalyzed for their ability to alleviate pain.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.